1. Field of the Invention
This invention relates to a method of selecting for high-expressing host cells, a method of producing a protein of interest in high yields and a method of producing eukaryotic cells having multiple copies of a sequence encoding a protein of interest.
2. Description of the Related Art
The discovery of methods for introducing DNA into living host cells in a functional form has provided the key to understanding many fundamental biological processes, and has made possible the production of important proteins and other molecules in commercially useful quantities.
Despite the general success of such gene transfer methods, several common problems exist that may limit the efficiency with which a gene encoding a desired protein can be introduced into and expressed in a host cell. One problem is knowing when the gene has been successfully transferred into recipient cells. A second problem is distinguishing between those cells that contain the gene and those that have survived the transfer procedures but do not contain the gene. A third problem is identifying and isolating those cells that contain the gene and that are expressing high levels of the protein encoded by the gene.
In general, the known methods for introducing genes into eukaryotic cells tend to be highly inefficient. Of the cells in a given culture, only a small proportion take up and express exogenously added DNA, and an even smaller proportion stably maintain that DNA.
Identification of those cells that have incorporated a product gene encoding a desired protein typically is achieved by introducing into the same cells another gene, commonly referred to as a selectable gene, that encodes a selectable marker. A selectable marker is a protein that is necessary for the growth or survival of a host cell under the particular culture conditions chosen, such as an enzyme that confers resistance to an antibiotic or other drug, or an enzyme that compensates for a metabolic or catabolic defect in the host cell. For example, selectable genes commonly used with eukaryotic cells include the genes for aminoglycoside phosphotransferase (APH), hygromycin phosphotransferase (hyg), dihydrofolate reductase (DHFR), thymidine kinase (tk), neomycin, puromycin, glutamine synthetase, and asparagine synthetase.
The method of identifying a host cell that has incorporated one gene on the basis of expression by the host cell of a second incorporated gene encoding a selectable marker is referred to as cotransfectation (or cotransfection). In that method, a gene encoding a desired polypeptide and a selection gene typically are introduced into the host cell simultaneously, although they may be introduced sequentially. In the case of simultaneous cotransfectation, the gene encoding the desired polypeptide and the selectable gene may be present on a single DNA molecule or on separate DNA molecules prior to being introduced into the host cells. Wigler et al., Cell, 16:777 (1979). Cells that have incorporated the gene encoding the desired polypeptide then are identified or isolated by culturing the cells under conditions that preferentially allow for the growth or survival of those cells that synthesize the selectable marker encoded by the selectable gene.
The level of expression of a gene introduced into a eukaryotic host cell depends on multiple factors, including gene copy number, efficiency of transcription, messenger RNA (mRNA) processing, stability, and translation efficiency. Accordingly, high level expression of a desired polypeptide typically will involve optimizing one or more of those factors.
For example, the level of protein production may be increased by covalently joining the coding sequence of the gene to a "strong" promoter or enhancer that will give high levels of transcription. Promoters and enhancers are nucleotide sequences that interact specifically with proteins in a host cell that are involved in transcription. Kriegler, Meth. Enzymol., 185:512 (1990); Maniatis et al., Science, 236:1237 (1987). Promoters are located upstream of the coding sequence of a gene and facilitate transcription of the gene by RNA polymerase. Among the eukaryotic promoters that have been identified as strong promoters for high-level expression are the SV40 early promoter, adenovirus major late promoter, mouse metallothionein-l promoter, Rous sarcoma virus long terminal repeat, and human cytomegalovirus immediate early promoter (CMV).
Enhancers stimulate transcription from a linked promoter. Unlike promoters, enhancers are active when placed downstream from the transcription initiation site or at considerable distances from the promoter, although in practice enhancers may overlap physically and functionally with promoters. For example, all of the strong promoters listed above also contain strong enhancers. Bendig, Genetic Engineering, 7:91 (Academic Press, 1988).
The level of protein production also may be increased by increasing the gene copy number in the host cell. One method for obtaining high gene copy number is to directly introduce into the host cell multiple copies of the gene, for example, by using a large molar excess of the product gene relative to the selectable gene during cotransfectation. Kaufman, Meth. Enzymol., 185:537 (1990). With this method, however, only a small proportion of the cotransfected cells will contain the product gene at high copy number. Furthermore, because no generally applicable, convenient method exists for distinguishing such cells from the majority of cells that contain fewer copies of the product gene, laborious and time-consuming screening methods typically are required to identify the desired high-copy number transfectants.
Another method for obtaining high gene copy number involves cloning the gene in a vector that is capable of replicating autonomously in the host cell. Examples of such vectors include mammalian expression vectors derived from Epstein-Barr virus or bovine papilloma virus, and yeast 2-micron plasmid vectors. Stephens & Hentschel, Biochem. J., 248:1 (1987); Yates et al, Nature, 313:812 (1985); Beggs, Genetic Engineering, 2:175 (Academic Press, 1981).
Yet another method for obtaining high gene copy number involves gene amplification in the host cell. Gene amplification occurs naturally in eukaryotic cells at a relatively low frequency. Schimke, J. Biol. Chem., 263:5989 (1988). However, gene amplification also may be induced, or at least selected for, by exposing host cells to appropriate selective pressure. For example, in many cases it is possible to introduce a product gene together with an amplifiable gene into a host cell and subsequently select for amplification of the marker gene by exposing the cotransfected cells to sequentially increasing concentrations of a selective agent. Typically the product gene will be coamplified with the marker gene under such conditions.
The most widely used amplifiable gene for that purpose is a DHFR gene, which encodes a dihydrofolate reductase enzyme. The selection agent used in conjunction with a DHFR gene is methotrexate (Mtx). A host cell is cotransfected with a product gene encoding a desired protein and a DHFR gene, and transfectants are identified by first culturing the cells in culture medium that contains Mtx. A suitable host cell when a wild-type DHFR gene is used is the Chinese Hamster Ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub & Chasin, Proc. Nat. Acad. Sci. U.S.A., 77:4216 (1980). The transfected cells then are exposed to successively higher amounts of Mtx. This leads to the synthesis of multiple copies of the DHFR gene, and concomitantly, multiple copies of the product gene. Schimke, J. Biol. Chem., 263:5989 (1988); Axel at al, U.S. Pat. No. 4,399,216; Axel et al., U.S. Pat. No. 4,634,665. Other references directed to cotransfection of a gene together with a genetic marker that allows for selection and subsequent amplification include Kaufman in Genetic Engineering, ed. J. Setlow (Plenum Press, New York), Vol. 9 (1987); Kaufman and Sharp, J. Mol. Biol., 159:601 (1982); Ringold et al., J. Mol. Appl. Genet., 1:165-175 (1981); Kaufman et al., Mol. Cell Biol., 5:1750-1759(1985); Kaetzel and Nilson, J. Biol. Chem., 263:6244-6251 (1988); Hung et al., Proc. Natl. Acad. Sci. U.S.A., 83:261-264 (1986); Kaufman et al, EMBO J., 6:87-93 (1987); Johnston and Kucey, Science, 242:1551-1554 (1988); Urlaub et al, Cell, 33:405-412 (1983).
To extend the DHFR amplification method to other cell types, a mutant DHFR gene that encodes a protein with reduced sensitivity to methotrexate may be used in conjunction with host cells that contain normal numbers of an endogenous wild-type DHFR gene. Simonsen and Levinson, Proc. Natl. Acad. Sci. U.S.A., 80:2495 (1983); Wigler et al., Proc. Natl. Acad. Sci. U.S.A., 77:3567-3570 (1980); Haber and Schimke, Somatic Cell Genetics, 8:499-508 (1982).
Alternatively, host cells may be co-transfected with the product gene, a DHFR gene, and a dominant selectable gene, such as a neo.sup.r gene. Kim and Wold, Cell, 42:129 (1985); Capon et al, U.S. Pat. No. 4,965, 199. Transfectants are identified by first culturing the cells in culture medium containing neomycin (or the related drug G418), and the transfectants so identified then are selected for amplification of the DHFR gene and the product gene by exposure to successively increasing amounts of Mtx.
As will be appreciated from this discussion, the selection of recombinant host cells that express high levels of a desired protein generally is a multi-step process. In the first step, initial transfectants are selected that have incorporated the product gene and the selectable gene. In subsequent steps, the initial transfectants are subject to further selection for high-level expression of the selectable gene and then random screening for high-level expression of the product gene. To identify cells expressing high levels of the desired protein, typically one must screen large numbers of transfectants. The majority of transfectants produce less than maximal levels of the desired protein. Further, Mtx resistance in DHFR transformants is at least partially conferred by varying degrees of gene amplification. Schimke, Cell, 37:705-713 (1984). The inadequacies of co-expression of the non-selected gene have been reported by Wold et al., Proc. Natl. Acad. Sci. U.S.A., 76:5684-5688 (1979). Instability of the amplified DNA is reported by Kaufman and Schimke, Mol. Cell Biol., 1:1069-1076 (1981); Haber and Schimke, Cell, 26:355-362 (1981); and Fedespiel et al, J. Biol. Chem., 259:9127-9140 (1984).
Several methods have been described for directly selecting such recombinant host cells in a single step. One strategy involves co-transfecting host cells with a product gene and a DHFR gene, and selecting those cells that express high levels of DHFR by directly culturing in medium containing a high concentration of Mtx. Many of the cells selected in that manner also express the co-transfected product gene at high levels. Page and Sydenham, Bio/Technolgy, 9:64 (1991). This method for single-step selection suffers from certain drawbacks that limit its usefulness. High-expressing cells obtained by direct culturing in medium containing a high level of a selection agent may have poor growth and stability characteristics, thus limiting their usefulness for long-term production processes. Page and Snyderman, Bio/Technology, 9:64 (1991). Single-step selection for high-level resistance to Mtx may produce cells with an altered, Mtx-resistant DHFR enzyme, or cells that have altered Mtx transport properties, rather than cells containing amplified genes. Haber et al, J. Biol. Chem., 256:9501 (1981);Assaraf and Schimke, Proc. Natl. Acad. Sci. U.S.A., 84:7154 (1987).
Another method involves the use of polycistronic mRNA expression vectors containing a product gene at the 5' end of the transcribed region and a selectable gene at the 3' end. Because translation of the selectable gene at the 3' end of the polycistronic mRNA is inefficient, such vectors exhibit preferential translation of the product gene and require high levels of polycistronic mRNA to survive selection. Kaufman, Meth. Enzymol., 185:487 (1990); Kaufman, Meth. Enzymol., 185:537 (1990); Kaufman et al., EMBO J., 6:187 (1987). Accordingly, cells expressing high levels of the desired protein product may be obtained in a single step by culturing the initial transfectants in medium containing a selection agent appropriate for use with the particular selectable gene. However, the utility of these vectors is variable because of the unpredictable influence of the upstream product reading frame on selectable marker translation and because the upstream reading frame sometimes becomes deleted during methotrexate amplification (Kaufman et al, J. Mol. Biol., 159:601-621 [1982]; Levinson, Methods in Enzymology, San Diego: Academic Press, Inc. [1990]). Later vectors incorporated an internal translation initiation site derived from members of the picornavirus family which is positioned between the product gene and the selectable gene (Pelletier et al, Nature, 334:320 [1988]; Jang et al, J. Virol., 63:1651 [1989]).
A third method for single-step selection involves use of a DNA construct with a selectable gene containing an intron within which is located a gene encoding the protein of interest. See U.S. Pat. No. 5,043,270 and Abrams et al., J. Biol. Chem., 264(24): 14016-14021 (1989). In yet another single-step selection method, host cells are co-transfected with an intron-modified selectable gene and a gene encoding the protein of interest. See WO 92/17566, published Oct. 15, 1992. The intron-modified gene is prepared by inserting into the transcribed region of a selectable gene an intron of such length that the intron is correctly spliced from the corresponding mRNA precursor at low efficiency, so that the amount of selectable marker produced from the intron-modified selectable gene is substantially less than that produced from the starting selectable gene. These vectors help to insure the integrity of the integrated DNA construct, but transcriptional linkage is not achieved as selectable gene and the protein gene are driven by separate promoters.
Other mammalian expression vectors that have single transcription units have been described. Retroviral vectors have been constructed (Cepko at al, Cell, 37:1053-1062 [1984]) in which a cDNA is inserted between the endogenous Moloney murine leukemia virus (M-MuLV) splice donor and splice acceptor sites which are followed by a neomycin resistance gene. This vector has been used to express a variety of gene products following retroviral infection of several cell types.
With the above drawbacks in mind, it is one object of the present invention to increase the level of homogeneity with regard to expression levels of stable clones transfected with a product gene of interest, by expressing a selectable marker (DHFR) and the protein of interest from a single promoter.
It is another object to provide a method for selecting stable, recombinant host cells that express high levels of a desired protein product, which method is rapid and convenient to perform, and reduces the numbers of transfected cells which need to be screened. Furthermore, it is an object to allow high levels of single and two unit polypeptides to be rapidly generated from clones or pools of stable host cell transfectants.
It is an additional object to provide expression vectors which bias for active integration events (i.e. have an increased tendency to generate transformants wherein the DNA construct is inserted into a region of the genome of the host cell which results in high level expression of the product gene) and can accommodate a variety of product genes without the need for modification.